Fluorescent membrane intercalating probes

ABSTRACT

The invention relates to a family of cyanine dyes which fluoresce in the far red and near infra red wavelengths of the spectrum and preferably possess lipophilic side chains. The dyes of the invention are soluble in commercially available membrane staining vehicles, are useful as probes for rapidly staining lipophilic structures such as membranes in cells or isolated from cells, and are well retained therein. Methods of using the dyes to detect stained cells both in vivo and in vitro are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation of application Ser. No.10/220,241 filed Nov. 12, 2002 now abandonded, which was the nationalstage of International Application No. PCT/US01/06923 filed Mar. 5,2001, which claims the benefit of application Ser. No. 60/186,682 filedMar. 3, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support undergrant number R43 CA86692 awarded by the National Cancer Institute, NCIand grant number R44 EB00228 awarded by the National Institute ofBiomedical Imaging and Bio Engineering, NBIB. Accordingly, the UnitedStates Government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to fluorescent, membrane intercalatingcompounds useful as dyes and probes. More particularly, the inventionrelates to lipophilic fluorescent compounds with an increased signal tonoise ratio that are useful for rapidly labeling a variety of lipophilicparticles or objects containing lipophilic structures, including cells,liposomes, microspheres and virus particles.

DESCRIPTION OF THE RELATED ART

It is known that fluorescent dyes have many uses and are particularlysuitable for biological applications in which the high sensitivitydetection of fluorescence is desirable. By binding to a specificbiological ingredient in a sample, a fluorescent dye can be used toindicate the presence or the quantity of the specific ingredient in asample. A variety of fluorescent dyes is available for fluorescentstaining and such dyes are employed in quantitation of, e.g., cells,proteins, DNA and RNA. Fluorescent dyes are also employed for monitoringcellular trafficking in response to various physiological conditions.Such dyes have a wide range of applicability in both clinical andresearch applications where cell sorting and monitoring of cellulartrafficking, proliferation and other responses are desired.

Fluorescent dyes are known to be particularly suitable for biologicalapplications in which a highly sensitive detection reagent is desirable.Dyes that are able to preferentially bind to a specific biologicalingredient or component in a sample enable the observer to determine thepresence, quantity or location of that specific ingredient or component.In addition, specific biological systems can be monitored with respectto their spatial and temporal distribution in diverse environments.Cyanines are particularly advantageous for such applications, due totheir high extinction coefficients and their amenability to thesystematic selection of structural variations which give predictableshifts in excitation and emission properties. As a result, cyanine dyeshave been used in various biological applications. The use of certaincationic lipophilic cyanine dyes, including DiIC₁₈, DiOC₁₈ and their C₁₂to C₂₂ homologs in combination with an osmolarity regulating agent tostain cells for the purposes of labeling viable cells, tracking stainedcells in vivo, and measuring cell growth rate has been previouslydescribed.

U.S. Pat. No. 4,762,701, which is incorporated herein by reference,refers to in vivo methods for tracking cyanine labeled cells thatfluoresce in the visible regions of the spectrum and for determiningcell lifetimes by measuring the rate at which the dye in labeled cellsadministered to a subject disappear.

U.S. Pat. No. 4,783,401, which is incorporated herein by reference,refers to methods for labeling viable cells with cyanine dyes thatfluoresce in the visible regions of the spectrum in order to, amongother things, measure the growth rate of cultured cells.

U.S. Pat. No. 4,859,584, which is incorporated herein by reference,refers to methods for determining the growth rate of cyanine labeledcells that fluoresce in the visible regions of the spectrum growing invitro and in vivo.

U.S. Pat. No. 5,804,389, which is incorporated herein by reference,refers to methods for determining abnormal cell shedding rates bylabeling cell membranes with cyanine dyes that fluoresce in the visibleregions of the spectrum and observing the rate at which the labeledcells are shed from the mucosal surface.

U.S. Pat. No. 6,004,536 to Leung et al., which is also incorporated byreference herein, refers to cyanine dyes possessing two lipophilic alkylchains that are preferably equal in length and incorporate either areactive functional group, or a phenyl, sulfo, sulfophenyl, or a bromoor chloro substituent that are useful for staining lipophilicstructures, such as membranes in cells or tissues, membranes isolatedfrom cells, natural or artificial liposomes, lipoproteins or polymers.Leung et al. states that the dyes are preferably soluble in an aqueousenvironment.

Flow cytometry and fluorescence activated sorting have been usedextensively to separate different classes of cells in the cellpopulations in blood and in bone marrow. Such methods have beenparticularly useful to separate the different types of leukocytes fromeach other, as a tool in typing of leukemias and lymphomas (See e.g.,U.S. Pat. No. 5,234,816), and to obtain blood stem cell progenitorfractions isolated away from other cell types (U.S. Pat. No. 5,137,809,Aug. 11, 1992), for research and for therapeutic uses. Flow cytometershave become routine in clinical laboratory use. Several parameters of acell may be measured simultaneously: forward scattered light is used tomeasure cell size; and a second scatter detector provides information onthe granularity of the cell cytoplasm. These methods can be used todifferentiate the various types of leukocytes. Fluorescent light emittedfrom various “fluorochromes,” each of which is bound to a specificcellular target molecule, is collected by the cytometer. Theseparameters create a broad range of applications dependent on thespecificity and combination of a dye-conjugated molecule and its target.

Although cytometry today relies upon correlated analysis of 3-4 colordata, the field is rapidly moving toward use of more probes/cell todissect complex inter and intracellular events by analyzing thecharacteristics of various subpopulations of cells in complex mixtures(as, e.g., in a developing immune response). The nature of excitationand emission characteristics of fluorochromes makes it difficult toselect more than three or four visible emitting fluorochromes attachableto cells which provide emissions sufficiently separated in wavelength togive good spatial and/or spectral discrimination.

General labeling of cell proteins or membranes with stable fluorescentprobes is also a powerful method for delineating intricate cell-cellinteractions, as for example when analyzing immune system functions.However, currently available protein and membrane labels, such as CFSE(Molecular Probes) and the PKH dyes (Sigma), have significantlimitations when studying cellular interactions and responses both invivo and in vitro. Because they excite and fluoresce in the visibleregions of the spectrum, high levels of tissue scattering andautofluorescence can render such dyes unsuitable for optical imaging inintact animals. In addition, cellular autofluorescence limits thesignal:noise (S/N) ratio that can be achieved and significant spectraloverlap with other commonly used visible fluors complicates instrumentsetup when such dyes are used for flow cytometry or confocal microscopy.Although longer wavelength analogs of DiO and DiI that are applicable togeneral membrane staining are known in the art, time as well asconcentration must be varied to achieve optimum staining with thesedyes.

The complex cell types, trafficking and localization patterns, signalingmechanisms, and regulatory feedback loops which constitute the innateimmune system allow it to respond highly selectively to a particularantigen or pathogen and also offer the potential to selectively enhanceor interfere with a response. However, this selectivity is achievedprimarily based on localized encounters involving antigen, antigenpresenting cells, and lymphocytes in the context of tissue specificadhesion molecules and secreted molecular messengers such as chemokinesand cytokines. Therefore, productive intervention in the immunesurveillance and response process requires the ability to dissect andmonitor complex cellular interactions in vitro, ex vivo, and in vivo.The ability to selectively tag different cell types and follow theirfate is critical to understanding immune responses in sufficient detailto design and optimize effective treatment strategies involvingimmunotherapy.

General membrane labeling with fluorescent lipophilic dyes whichintercalate stably into cell membranes is simple, rapid, and applicableto almost any cell type. Currently available probes of this type havebeen utilized for purposes of tracking and identifying specific celltypes and they offer several advantages in contrast to utilizing generalprotein labeling for such purposes. Since labeling is non-covalent andoccurs by partitioning into the lipid bilayer, there is no waitingperiod for fluorescent intensity to stabilize, such as is required forcovalent protein labels (e,g., CFSE), and untoward effects on cellularreceptor-ligand interactions and associated responses are typicallyminimal.

The most common fluorophores used to label cells and biomolecules wereoriginally developed for microscopy, and for reasons of compatibilitywith available light sources and the human eye, fluoresce primarily inthe UV and visible regions of the spectrum (approximately 400 to 600nm). Dilution of membrane intercalating dyes among daughter cells hasproven very useful for monitoring differential cell proliferationresponses in complex populations and for tracking of cells in respondingto stimuli such as antigenic challenge. Like general protein labels,concentration of membrane dyes is halved with each cell division, thuslimiting use for long term tracking. Also, in both general proteinlabeling and fluorescent membrane labeling, high labeling intensity(often 1-2 orders of magnitude greater than bright antibody labeling)can complicate filter selection and color compensation when used incombination with other probes.

The above challenges and limitations to fluorescent labeling havebrought increasing interest in development of fluors that excite andemit in the FR (far red) and NIR (near infrared) wavelengths. Althoughusage in the literature varies considerably, we here define FR as about600-700 nm and NIR as about 700-900 nm, since water absorption andthermal background begin to interfere with measurement of biologicalfluorescence at >900-1000 nm. The use of FR or NIR fluors has a numberof significant advantages in biological systems in general, and forcellular analysis in particular. These include i) decreased backgroundcaused by tissue or protein autofluorescence, ii) decreased backgroundcaused by Raman scatter, iii) less spectral overlap when used inconjunction with common UV or visible fluors, and iv) excitation andemission profiles compatible with the use of inexpensive excitationsources (e.g. diode lasers) and detectors (e.g. avalanche diodes). FRfluorescing analogs can be used on existing flow cytometers and confocalmicroscopes, since many of these instruments have FR excitationcapability (HeNe, 635 nm diode, or 647 nm Kr/Ar lasers). Use of FRanalogs therefore provides i) ability to do longer term in vitro and invivo tracking of dividing cells due to reduced background and improvedsignal/noise ratio and ii) simpler instrument setup due to reducedspectral overlap.

Utilization of NIR fluorescence has significant advantages over even FRimaging for in vivo optical imaging of intact tissues or animals. Inaddition to decreased background from autofluorescence and Ramanscattering, NIR light is better transmitted in vivo and thus real timefluorescence imaging can be performed through millimeters to centimetersof tissue. In fact, the longer the wavelength of the exciting light orthe NIR flourescence, the better the tissue penetration, due to reducedelastic scattering and the fact that the few biomolecules which absorbin this region (hemoglobin and deoxyhemoglobin) do so only weakly. FRand NIR labeled antibodies or polymers have been shown to enhancecontrast between normal tissue and tumors. Current depth of detection isin the 0.5-1 cm range but NR light can travel through tissue for 5-6 cm.

It is known that adjusting the composition of aromatic groups and thenumber of methine groups separating the aromatic groups of cyanine dyescauses changes in the light excitation and emission patterns and colorof these dyes. In general, increasing the number of methine groupsseparating aromatic components of the dyes will shift the emissionspectra toward the red and near infrared wavelengths. Increasing thelength of the methine bridge between aromatic groups, however, alsoincreases the overall liphophilicity of the compound and thus willreduce the solubility of such compounds, limiting their utility asmembrane probes. This limitation can be overcome if appropriatecompositions for labeling are selected which afford sufficient aqueoussolubility to be compatible with sensitive biological materials (e.g.,cells) while minimizing the negative impact of standard physiologicalmedia and salts on the lipophilic substances.

SUMMARY OF THE INVENTION

Accordingly, the present invention is directed to novel membraneintercalating dyes (also referred to herein as “probes”) that fluorescein the far red and near infrared segments of the spectrum and aresoluble in commercially available membrane staining diluents. Theseprobes are useful in diagnostic and therapeutic applications. Thepresent invention is also directed to compositions containing the probesin pharmaceutically acceptable aqueous and non-aqueous labeling vehiclessuitable for staining biological materials.

In another aspect, the invention provides methods of contacting theprobes with cells and/or other lipophilic structures, allowing theprobes to intercalate with the lipophilic structures, and detecting thecells or other lipophilic structures in vitro and or in vivo based onthe fluorescence emitted therefrom.

In a preferred embodiment, the invention is directed to method oflabeling epithelial cells in vivo with cyanine dyes to provide an invivo method for diagnosing disease states which are characterized by thepresence of abnormal cell shedding rates amongst mature epithelialcells.

To achieve these and other objects, the present invention provides an invivo method for detecting abnormal cell shedding rates amongst matureepithelial cells, such as epithelial cells of mucosal surfaces, of awarm-blooded animal comprising the steps of labelling mature surfaceepithelial cells at a target site with FR and NIR probes and thereaftermonitoring the site for the presence or absence of the label. In apreferred embodiment of this method, the cells which are labeled resideon mucosal surfaces; amongst which mucosal surfaces of thegastrointestinal tract provide particularly preferred targets. Thepresent invention also provides a method for diagnosing disease statescharacterized by abnormal cell shedding rates amongst mature epithelialcells of a warm-blooded animal, comprising labeling mature epithelialcells with the FR and NIR probes of the invention, determining theshedding rate of the labeled-cells and comparing the shedding rate ofthe labeled cells to the known shedding rate of similarly locatedhealthy epithelial cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the presently preferredembodiments of the invention. The invention provides compounds that arenovel membrane intercalating dyes (also referred to herein as “probes”)that fluoresce in the far red and near infrared segments of the spectrumand methods for their use. The invention also provides labelingcompositions comprising the probes solubilized in a labeling vehicle.The probes are solubilized in a vehicle compatible with the biologicaland/or solvent sensitivities of the materials to be labeled.

The compounds contain lipophilic tails (referred to herein in thestructures as “R” and “R′”) that in some embodiments are equal in lengthwhile in some preferred embodiments, R≠R′.

It is contemplated that the probes of the present invention may be usedin conjunction with other labeling techniques and reagents formultiparameter cell tracking and sorting procedures. Usefulness of a dyein combination with others is traditionally determined by two aspects ofspectra of light energy interactions: (1) the extent to which a dyemolecule is excited by a single illumination wavelength or narrow bandof wavelengths and (2) the extent to which each excited dye moleculeemits light of wavelength sufficiently different from the other dyes soas to be discernible as a unique color or peak. The first aspect enablesthe user to illuminate the multiply-stained biological sample with asingle wavelength and the second aspect enables the user to observe andrecord different colors of emission, each of which is associated with aparticular cell type or a structure.

There are a number of new detectors under development in which theentire spectrum of light is collected and then the curves characteristicof each probe's emission are devolved from the aggregate signal takenover the entire spectrum. It is also contemplated that the probes of thepresent invention will be readily detected using such instruments.

The probes of the invention, when used as membrane-intercalating dyesselectively stain cell membranes in vitro, ex vivo and in vivo and donot undesirably affect the nature of the cells. The probes and thus thecells or other lipophilic structures to which they become attached canbe readily identified by the fluorescence they emit. In addition, thecompounds of the present invention are not cytotoxic when used atappropriate concentrations, are stably retained in cell membranes, andstain cells rapidly. The probes need only be applied to cells for a fewminutes to achieve staining intensities 100-1000 times greater thanbackground autofluoresence. Another beneficial-aspect of the compoundsof the present invention is that they are soluble in isotonic salt freediluents suitable for membrane labeling such as Diluent C (Sigma-AldrichCorporation). The probes of the present invention are useful generallyas agents for cell labeling, cell sorting and cell tracking in vitro andin vivo for both basic (laboratory) and clinical research. For examplesof such uses, see U.S. Pat. Nos. 5,385,822; 5,256,532, the disclosuresof which are incorporated by reference herein; and U.S. Pat. Nos.4,859,584; 4,783,401 and 4,762,701. Such probes will be useful, forexample, as research reagents for use in existing flow cytometers andconfocal imaging systems which have FR and/or NIR capabilities.

The probes of the present invention will 1) bind to cells in sufficientnumber to give a good signal compared to autofluorescence; 2) not betoxic to the cells at that level, and 3) be retained in the cellmembrane long enough for tracking and/or sorting of particular subgroupsof cells to be completed.

The invention provides probes that exhibit<5% change in HPLC purity forat least 2 months in solid form when stored at room temperature and atleast I month in ethanol and maintain>60% of their initial solubility inan ethanol control for at least 30 minutes in an aqueous vehicle.

The FR/NIR probes of the invention exhibit<10% photobleaching after 24hr under ambient lighting typical to indoor fluorescent lighting.

The FR probes of the invention result in<10% reduction in cell viabilityand<10% alteration in the population doubling time of cultured YAClymphoma cells after labeling with concentrations sufficient to givestarting S/N ratios>100.

The NM probes of the invention result in<10% reduction in cell viabilityand<10% alteration in the population doubling time of cultured YAClymphoma cells after labeling with concentrations sufficient to givestarting S/N ratios>10.

The FR probes of the invention are retained in cellular membranes wellenough to maintain an S/N>100 after 24 hrs co-culture with unlabeledcells even when culturing is carried out under conditions where dyeintensity is decreased by cell growth.

The NIR probes of the invention are retained in cellular membranes wellenough to maintain an S/N>10 after 24 hrs co-culture with unlabeledcells under conditions where dye intensity is decreased by cell growth.

The FR and NIR probes of the invention require<40% correction forspectral overlap in the phycoerythrin channel, as evaluated by flowcytometry.

The lipophilic nature of the probes of the present invention providesfor the efficient incorporation of the probes into various lipidcontaining or hydrophobic structures, including cell and viralmembranes, liposomes, microspheres and the like. When incorporating theprobes into cells and virions, a labeling composition comprising theprobe and an aqueous labeling vehicle is mixed with the target to belabeled. The labeling composition contains a cyanine dye in a vehicle(diluent) that is safe for application and that provides reproduciblecell labeling. Osmolarity regulating agents in which cyanine dyes formstable solutions for at least as long as required for labeling can beused. Acceptable osmolarity regulating agents may be selected fromsugars including monosaccharides such as glucose, fructose, sorbose,xylose, ribose, and disaccharides such as sucrose; sugar-alcoholsincluding mannitol, glycerol, inositol, xylitol, and adonitol; aminoacids including glycine and arginine; and certain Good's buffers such asN-tris(hydroxymethyl)-methyl-3-aminopropanesulfonic acid. Small amountsof buffering agents may be added to the labeling medium to regulatehydrogen ion concentration (pH) to physiological and/or nontoxic levels.Other conventional agents, such as antibiotics and preservatives, may bealso be employed in the vehicle, but only to the extent that they do notcreate salt concentrations that induce rapid formation of dye micellesor aggregates.

When incorporating the probes into microspheres or liposomes and likematerials which can tolerate exposure to non-aqueous labeling vehicleswithout detrimental effect, a labeling composition comprising the probeand a non aqueous labeling vehicle is mixed with the target to belabeled. Non aqueous labeling vehicles include polar organic solventssuch as ethanol, dimethyl formamide, dimethylsulfoxide, and the like.

Despite their lipophilic nature, we have found that the probes of thepresent invention are sufficiently soluble in aqueous vehicles to allowefficient and rapid staining of liphophilic structures (membranes andthe like) which are detrimentally affected by exposure to polar organicsolvents.

The detection step can employ a luminescence microscope or other opticalimaging apparatus such as e.g., a fiber optic diagnostic device such asa cystoscope or endoscope and the like, having a filter for absorptionof scattered light of the excitation wavelength and for passing thewavelength that corresponds to the fluorescence corresponding to theparticular dye label used with the specimen. Preferred methods ofobservation and analysis include direct visualization with a microscopefitted with a light source and filters appropriate to the excitation andemission wavelengths, and use of a camera attached to the microscopes.

A preferred method of the invention for cell separation and enumerationof live cells appropriately stained with this class of probe reagents,is isolation by use of flow cytometry apparatus such as by way ofexamples only a FACSCalibur instrument with sorting capability or aFACSVantage instrument with cell sorting capability. Other similarinstruments are well known in the art. These instruments illuminate amixed cell population, for example at a given wavelength with an argonlaser source of light, and use an emission signal from each celldetected in a moving fluid such as a buffer, to sort each cell as it isflowing past the detector using a variety of filters for collection ofemitted light using techniques well known in the art. The apparatus cancount and/or collect cell populations based on calculations employingmeasures of emitted right yielding both data and cell fractions forfurther analysis and use.

Recent advances in technology have made it possible to do fluorescenceimaging not only at the microscopic (cellular and subcellular) level butalso at the macroscopic (whole tissue or whole body) level. The probesof the present invention provide useful solutions to the problem ofspectral “pollution” or spill, which is frequently encountered whendoing multiprobe studies using confocal imaging or other quantitativemicroscopy techniques. In addition, the NIR probes, when combined withmacroscopic imaging methods, provide a useful alternative to radiolabelsfor monitoring immune cell trafficking, localization and redistributionin intact animals as well as potential for use in diagnosis and/orphototherapy.

In another preferred embodiment, the probes of the present inventionare, a subject for detection of abnormal epithelial cell shedding ratesin vivo such, as described in U.S. Pat. No. 5,804,389. In this process,the abnormal shedding of epithelial cells in a warm-blooded animal isdetermined. The process includes the steps of labeling mature surfaceepithelial cells with the compounds of the present invention at a targetsite, exposing the cells to light of an excitation wavelength andthereafter monitoring the site for the presence or absence of the label,and observing the loss of detectable label over a pre-defined period oftime. The present invention also provides a method for diagnosingdisease states characterized by such abnormal cell shedding ratesamongst mature epithelial cells of a warm-blooded animal, comprisinglabeling mature epithelial cells, determining the shedding rate of thelabeled cells and comparing the shedding rate of the labeled cells tothe known shedding rate of similarly located healthy epithelial cells.

The probes of the present invention can be utilized to determineabnormal shedding rates on any epithelial surface of the body. Surfacesinclude those of the stomach, biliary tract, colon, urinary tract-bloodvessels, pulmonary tract-including the nasal cavity, cornea, esophagus,pancreatic duct, small intestine, and genital organs including thevagina and ovarian duct and the prostate gland. Preferably, thecomposition in solution form is administered by direct application (e.g.spraying) onto the surface of the epithelial mucosa under direct visionby endoscope, by flooding the surface with a solution, or by orallyadministering the solution to the subject in the form of a drink.

When in vivo use in humans is contemplated, a solution of any of thecompounds of the present invention can be prepared by dissolving aneffective amount of the probe in an isoosmotic, aqueous and preferablysalt-free solvent miscible with both water and polar organic solvents.The concentration levels of dyes in compositions for in vivo useaccording to the present invention will be similar to or greater thanthe concentration levels used in the previously-known in vitro cellstaining applications of those dyes. The precise concentration to beadministered can be varied and can be readily optimized. The volume ofprobe composition to be administered will vary depending upon theconcentration of the cyanine dye in the composition and upon the size ofthe target site. The administration volume may vary, for example, fromabout 1 to 100 ml and can be readily optimized. An administration volumeof about 10 nil of the probe composition can be used in manyapplications. Administration route will vary depending upon the type ofcells to be labeled. As indicated above, for staining of epithelialcells may be best achieved by oral administration or direct application.Other modes of administration, such as subcutaneous, intramuscular andintravenous injection and the like are also contemplated.

According to the preferred method of the invention, the cyanine dyelabel is detected by exposing the site of labeled cells to excitationlight and observing and/or measuring the intensity of the fluorescence.For example, compound 8 (discussed infra in the examples) responds toexcitation light of about 635 nm for the observation of maximumfluorescence at 667 nm, whereas compound 13 (also discussed infra in theexamples) responds to excitation light of about 647 nm for theobservation of maximum fluorescence at 713 mm.

The probes of this invention can be synthesized, as set forth in detailin the examples below.

The FR probes of the present invention can be detected by commercialcytometry instrumentation with the ability to excite fluors which absorbin the 600-700 mm range and emit in the 700-800 range (e.g., BDFACSVantage, FACSCalibur, Beckman Coulter Altra, and Cytomation MoFloflow cytometer/sorters, and BioRad 1024ES or 1024MP confocal imagingsystems). The advent of small benchtop cytometers (e.g. volume capillarycytometer in development by Biometric Imaging/BD which incorporateinexpensive diode lasers and diode detection systems will provideadditional detection systems to quantify NIR fluorescence.

This near infrared/far red wavelength also is advantageous in that thebackground fluorescence in this region normally is low in biologicalsystems and high sensitivity can be achieved.

A preferred labeling composition of the present invention comprises acyanine dye of the formula:[A-CR₁═CR₂—Y═CR₃—CR₄═B]_(Z) ⁻ ⁺wherein “—Y═”is selected from the group consisting of—CR₅═, —CR₆═CR₇—CR₈═, —CR₉═CR₁₀—CR₁₁═CR₁₂—CR₁₃═, and

wherein each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, andR₁₃ is independently H, halogen or an alkyl group having 1-4 carbons;X is selected from the group consisting of H, halogen, O-alkyl, O-aryl,S-alkyl and S-aryl;Z is a biologically compatible counterion;“A-”is a structure selected from the group consisting of

and “═B”is selected from the group consisting of

wherein D and E are each independently O, S or CR₁₄ R₁₅, where R₁₄ andR₁₅, which may be the same or different, are independently alkyl groupshaving 1-6 carbons. Alternatively, R₁₄ R₁₅ taker in combination completea 5- or 6-membered saturated ring. Preferably X equals Y (yielding asymmetrical cyanine). In some preferred embodiments, both D and E areCR₁₄ R₁₅, where CR₁₄ R₁₅ are methyl or ethyl, more typically methyl.Each of R₁₆-R₄₇ is independently H, halogen or an alkyl group of 1-4carbons.

R and R′ are each independently linear or branched hydrocarbons having7-30 carbons, with the proviso that one of R and R′ must be at least 14carbons.

Z is a biologically compatible counterion that is typically an anionthat balances the intrinsic positive charge of the cyanine dye and ispresent in such a number and with such a total charge as to make theoverall molecule electrically neutral. As used herein, a substance thatis biologically compatible is not toxic as used, and does not have asubstantially deleterious effect on biomolecules. Such compounds alsomust not undesirably affect cell viability in the concentrationsrequired for labeling. Accordingly, pharmaceutically acceptable forms ofthe cyanine dye other than the iodide salt may be employed in someinstances, including other pharmaceutically acceptable salts. Examplesof useful counterions for dyes having a net positive charge include, butare not limited to, chloride, bromide, iodide, sulfate, alkanesulfonate,arylsulfonate, phosphate, perchlorate, tetrafluoroborate, nitrate andanions of aromatic or aliphatic carboxylic acids. Selection of anappropriate anion, however will be limited by the particular anions'affect on solubility, since it is known that the anion associated withthe various lipophilic molecules can effect the solubility of thecompound.

In a preferred embodiment, the counter ion is iodide. In certaininstances, such as for in vivo administration of the probes, it may bepreferable to substitute the iodide counterion with a chloride, sincesome individuals are allergic to iodine.

The present invention is further described in the following exampleswhich are provided for illustrative purposes only and are not to beconstrued as limiting. Standard techniques well known in the art or thetechniques specifically described below are utilized.

EXAMPLE 1 Synthesis of Probe with 667 nm Emission (8)

A membrane-probe with 667 nm Emission was prepared according to thefollowing synthetic reaction Scheme 1.

Preparation of 1-tetradecyl-2,3,3-trimethylindolinium Iodide (compound5)

Tetradecyl-1-(4-chlorobenzenesulfonate): To a stirred solution oftetradecanol (26.3 g, 0.123 mol, Aldrich Chemical Co., Milwaukee, Wis.)and 4-chlorobenzenesulphonyl chloride (28.48 g, 0.123 mol, Aldrich) indichloromethane (500 ml) at room temperature is added triethylamine (28ml, Aldrich) in dichloromethane (200 ml) dropwise. The resultingsolution is stirred for 48 hours. The reaction mixture is then washedwith water (3×400 ml) and the dichloromethane layer dried over sodiumsulfate and concenetrated. The crude solid obtained was recrystallizedfrom methanol to provide puretetradecyl-1-(4-chlorobenzenesulphonate)(29.9 g, 62%) as a white solid.200 MHz proton NMR (CDCl₃): 0.88 (t, J=7.0 Hz, 3H), 1.10-1.80 (m, 24H),4.05 (t, J=7.0 Hz, 2H), 7.50-7.60 (m, 2H), 7.80-7.90 (m, 2H).

2,3,3-trimethyl-(3H)-indoleine (4) (6.36 g, 0.04 mol, Aldrich) andtetradecyl-1-(4-chlorobenzenesulfonate) (15.52 g, 0.04 mol) are heatedtogether with stirring at 130-140° C. for 3 h. The reaction mixture isthen cooled to room temperature and dissolved in ethanol (200 ml). Asaturated solution of potassium iodide (200 ml) is added and thismixture is stirred for 30 minutes 1.5 L of distilled water is then addedand after a further 15 minutes stirring, the solid precipitate iscollected, washed with water and dried under vacuum. The crude materialis recrystallized from ethyl acetate to provide pure intermediatecompound 5 (11.9 g, 62%). 300 MHz proton NMR (CDCl₃): 0.88 (t, J=7 Hz,3H), 1.25-1.50 (m), 1.67 (s, 6H), 1.93 (m, 21), 3.13 (s, 3H), 4.70 (t,J=7.70 Hz, 2H), 7.60-7.66 (m, 4H).

Preparation of 1-docosanyl-2,3,3-trimethylindolinium Iodide (6)

Docosanyl-1-(4-chlorobenzenesulfonate): To a stirred solution ofdocosanol (47.8 g, 0.15 mol) and 4-chlorobenzenesulphonyl chloride(34.01 g, 0.16 ml, Aldrich) in dichloromethane (500 ml) at roomtemperature is added triethylamine (33.5 ml, Aldrich) in dichloromethane(200 ml) dropwise. The resulting solution is stirred for 48 h. Thereaction mixture is then washed with water (3×400 ml), the organic layerdried over sodium sulfate and concentrated to˜400 ml to initiatecrystallization of the product. After cooling and aging the precipitateis collected by filtration and dried under vacuum to provide puredocosanyl-1-(4-chlorobenzenesulphonate) (48.9 g, 73%) as a white solid.300 MHz proton nmr (CDCl₃): 0.88 (t, J=7.0 Hz, 3H), 1.15-1.40 (m),1.55-1.75 (m, 2H), 4.02 (t, J=7.0 Hz, 2H), 7.48-7.60 (m, 2H), 7.78-7.90(m, 2H).

2,3,3-trimethyl-(3H) indoleine (6.3 g, 0.04 mol, Aldrich) anddocosanyl-1-(4-chicrobenzenesulfonate) (20.0 g, 0.04 mol) are heatedtogether with stirring at 130-140° C. for 3 h.

The reaction mixture is then cooled to room temperature and the waxysolid dissolved in ethanol (250 ml). A saturated solution of potassiumiodide (200 ml) is, added and this mixture stirred for 30 minutes 1.0 Lof distilled water is then added and after a further 15 minutesstirring, the solid precipitate is collected, washed with water anddried under vacuum. The crude material is recrystallized fromdichloromethane/hexane to provide pure intermediate compound 6 (14.5 g,61%). 200 MHz proton NMR (CDCl₃): 0.87 (t, J=7.0 Hz, 3H), 1.15-1.50 (m),1.67 (s, 6H), 1.85-2.00 (m, 2H), 3.10 (s, 3H), 4.70 (t, J=7.7 Hz, 2H),7.55-7.70 (m, 4H).

Preparation of1-docosanyl-2-[(4-N-phenyl-N-acetylamino)-1,3-butadienyll-3,3-dimethylindolinilimiodide (7)

A solution of (6) (2.38 g, 4-mmol) and malonaldehyde bisphenyliminehydrochloride (1.10 g, 4.4 mmol, TCI America, Portland, Oreg.) in aceticanhydride (30 ml) is heated at 100-110° C. for 1 h, cooled to roomtemperature and filtered. The filtrate is then diluted with 300 ml ofwater and placed in a refrigerator at 0-5° C. for 24 h. The resultingprecipitate is collected and dried under vacuum to provide (7) (0.65 g,21%). 300M proton NMR (CDCl₃): 0.88 (t, J=6.5 Hz, 3H), 1.20-1.40 (m),1.70 (m), 1.80 (s, 6H), 2.24 (s, 3H), 4.36 (t, J=7.5 Hz, 2H), 5.79 (t,J=12.8 Hz, 1H), 6.71 (d, J=15.0 Hz, 1H), 7.25-7.70 (m, 9H).

Intermediate compound 7 (0.544 g, 0.71 mmol) and intermediate 5 (0.343g, 0.71 mmol) are heated together in refluxing dichloromethane (10 ml)containing 10 drops of triethylamine for 3 h. The reaction is monitoredby TLC (5% methanol in dichloromethane). The reaction mixture is thenconcentrated by rotary evaporation and the residue crystallized frommethanol at −20° C. overnight. The solid is collected and purifiedfurther by silica gel flash column chromatography eluting with 5-7.5%methanol in dichloromethane. Pure fractions are combined andconcentrated to provide compound 8 (210 mgs, 30%). Purity>97% by HPLC.300 MHz proton NMR (CDCl₃): 0.88 (t, J=6.3 Hz, 6H), 1.25-1.55 (m), 1.66(m), 1.81 (s, 16H), 4.04 (t, J=7.4 Hz, 4H), 6.29 (d, J=13.6 Hz, 2H),6.78 (t, J=12.4 Hz, IH), 7.05 (d, J=7.8 Hz, 1 Hh.), 7.20-7.70 (m, 2H),7.33-7.38 (m, 4H), 8.32 (t, J=13.0 Hz, 2H). Electrospray massspectroscopy. M′=860. UV/VIS (ethanol): λmax=648 nm, c=189,270M-1 cm-1.Fluorescence (ethanol): excitation max.=647 nm, emission max.=667 nm.

A probe with this emission wavelength was selected for synthesis becauseit is red shifted by>100 nm compared with most other visible fluorescentprobes used for cell labeling and therefore has minimum spectral overlapwith them. This probe could therefore be ideal for use in flow cytometryand confocal microscopy when multiprobe labeling is required.

EXAMPLE 2 Synthesis of FR Probe with 713 nm Emission (13)

This compound prepared from compound 9 (available from Fisher/AcrosChemicals) as shown in Scheme 2 below using similar types of reactionsand conditions to those described for compound (8).

EXAMPLE 3 Synthesis of FR Probe with 682 nm Emission (25)

This compound was synthesized by coupling of intermediate 6 fromreaction scheme 1 with intermediate 10 of reaction scheme 2 understandard conditions. The compound was purified by column chromatographyusing standard conditions. This compound provides a far red emittingprobe with absorbance and fluorescence properties intermediate tocompound (8) and compound 13. The final isolated yield for this productwas slightly lower (11.5%) than for

Compound (8) and compound (13) due to difficulty in removing thesymmetrical byproduct formed by dimerization of intermediate 7.

EXAMPLE 4 Synthesis of NIR Probe with 814 nm Emission (15)

This compound is prepared as shown in Scheme 3 below, using similartypes of reactions and conditions to those already described above. Thedesired probe is purified by recrystallization or chromatography.

EXAMPLE 5a Synthesis of NIR Probes with 837 nm Emission (19) and (26)

Compound 19 is prepared as shown in Scheme 4a.

Compound 17 is prepared in ˜27% yield according to procedures known inthe art. Heating compound (11) with Compound 17 furnishes intermediatecompound 18 in good yield. Reaction of Compound 18 with Compound 10under the standard base coupling conditions will provide Compound 19.This probe is closely matched with commercially available diode laserswith emission wavelength of 810 mm. Excitation at wavelengths above 800mm are to be used when labeling structures that are observed atrelatively greater tissue depths, due to reduced light absorption byhemoglobin and myoglobin at these wavelengths, while maintaining theadvantages of decreased scattering and autofluorescence associated withNIR illumination in general. As with compound 15, cells labeled withcompound 26 should be readily detected using techniques well known inthe art.

The optimal conditions for coupling of 17 with 2 mole equivalents of 11is reflux in ethanol containing sodium acetate. The resulting compoundis treated with potassium iodide and purified by silica gelchromatography.

EXAMPLE 6 Synthesis of a NIR Probe with 900 nm Emission (24)

Excitation and emission further into the IR can be desirable to increasetissue penetration and depth of strutures detectable using macrosopicimaging. This compound is prepared as shown in Scheme 5, using reactionssimilar to those described above.

EXAMPLE 7 Absorbance/Emission Spectra

Referring now to Table 1 there are shown representations of theabsorbance and emission properties of some example compounds of thepresent invention. Absorption and Fluorescence spectra were nm using0.25 μM solutions in ethanol on a Jasco, Inc. UV-530 UV/VISspectrophotometer and FP 750 spectrofluorometer, respectively.Approximate extinction coefficients were estimated from the absorbancescans by dividing the maximum absorbance by the nominal concentration(25 μM).

TABLE 1 Compound Absorbance Max Excitation Max Emission Max 8 626 647667 13 684 680 713 15 786 790 814 25 666 667 682 26 824 824 837

EXAMPLE 8 Chemical Stability of FR/NIR Probes

Chemical stability of example compounds of the present invention whenstored at room temperature as a solid or as a 1 mM solution in ethanolwas determined by HPLC. Integration of the peaks detected for examplecompounds of the present invention at 260 nm was used to estimatechemical stability. All compounds tested displayed<5% change in HPLCpurity when held as a solid at room temperature for two months and whenheld as a 1 mM liquid solution in absolute ethanol at room temperaturefor one month.

EXAMPLE 9 Photostability of FR/NIR Probes

1 mM solutions of the indicated probes in absolute ethanol were placedunder normal fluorescent room lighting. Aliquots were taken from eachsample shortly after preparation and the absorbance measured at theabsorbance maximum for each probe as in Example 6. The absorbance ofeach sample was again measured after 24 hours at room temperature underambient fluorescent light. All probes were stable, exhibiting<5%photobleaching for each compound.

EXAMPLE 10 Solubility of FR/NIR Probes

Effects of the various combinations of asymmetrical and symmetricalalkyl chains and methene bridges on the solubility of the compounds ofthe present invention were examined. Compounds 8, 25, 13, 15 and 26 wereall soluble in ethanol at concentrations≧1 mM. The more lipophilicprobes 15 and 26, however, required sonication for a few minutes toachieve dissolution at 1 mM in ethanol. All example probes-were poorlysoluble in water. Triplicate 1:50 dilutions of stock 1 mM probesolutions were made in either ethanol or test diluent. After 30 minutesat room temperature solutions were centrifuged at 10,000×g for 10minutes to remove any microagregates and 100 ml aliquots weretransferred to 2.0 ml of ethanol for absorbance determinations. Based onthe teachings of Leung et al. and on solubility specifications found onSigma Certificates of Analysis for the commercial diluents (diluentsupernatant absorbance at 30 minutes at least 60% of ethanol supernatantabsorbance after 30 minutes), a preliminary specification at least 60%of the ethanol control was set as a surrogate for solubility sufficientto achieve cell labeling with S/N ratios greater than 100 and tominimize potential problems with brightness or reproducibility oflabeling. None of the probes was as soluble after 30 minutes in DiluentC as PKH26 (86%±4% of ethanol control). We have found, however, that atleast for compound 8, 25 and 13, cells stained using compositions of dyein Diluent C gave S/N ratios substantially greater than those for PKH26(Table 2), indicating that solubility was not a limiting factor for celllabeling. After 30 minutes, compounds 8 and 15 retained approximately65% of their solubility at T0, compounds 13 and 25 greater than 55% oftheir solubility at T0, and compound 26 retained approximately 20% ofits solubility in Diluent C at T0. This example demonstrates that theaddition of polar groups or other substituents to enhance watersolubility as taught by Leung et al. is not an essential prerequisitefor bright and reproducible cell labeling with these-agents. Inaddition, it shows that useful levels of cell labeling can be obtainedeven when solubilities in iso-osmotic diluents are less than thosetaught by Horan et al.

EXAMPLE 11 Characterization of FR and NIR Probe Effects on CellViability, Cell Growth, and S/N Ratios

YAC-1 murine lymphoma cells were stained at a concentrations of 10⁷cells/ml with increasing concentrations of probe by rapidly admixing 2×cells suspended in Diluent C with 2× probe in Diluent C. After 5 minutesat room temperature, the staining was stopped by the addition of cellculture medium. Cells were then washed thoroughly (RPI1640+10.0% FBS)and cultured under standard conditions for 22-24 hours. Cell numberswere compared to those in a control culture treated with diluent alone.

Maximum Tolerated Concentration (MTC) of probe was defined as thehighest concentration of probe at which (1) viability by trypan blueexclusion immediately post-staining was>90% and (2) YAC cell growthafter 24 hours was no more than 10% different than growth of cells indiluent alone.

For S/N and spectral crossover determinations, cells were labeled inDiluent C using the MTC for each probe and then co-cultured with anequal number of unlabeled cells treated with diluent only. An aliquot ofcells was fixed with 1% buffered methanol free formalin immediatelyafter staining (T0) and analyzed on a flow cytometer. PMT high voltageon the flow cytometer was set so that the entire population of unlabeledcells lay in the first decade of the 4 decade log intensity scale,thereby allowing determination of a true mean value for unlabeled cells.When the intensity of stained cells was so great that the high voltagesetting used for analysis caused a significant number of unlabeled cellsto fall above the 4th decade, the high voltage setting was reduced tobring labeled cells fully on scale. Multilevel Rainbow beads fromSpherotech (Libertyville, Ill.) were then run at both PMT settings andused to calculate adjusted intensity values for labeled cells based onthe shift in intensity for the brightest bead peak. Results of viabilityare summarized in Table 2 below.

TABLE 2 Growth Inhibi- S/N at % Retention at Compound MTC Cytotoxicitytion T = 0 T = 22 hours Target <10% at <10% >100 >90% (growth- CriteriaT = 0 for FR corrected) >10 for NIR PKH26 10 μM  <1% <1%  265 106%  357compound 10 μM  <1% <1% 3948  98% 8 compound 10 μM  <1% <4% 1031  91% 25compound 10 μM  <1% <1% 1152  90% 13 compound 10 μM  <1% <5% NC* NC 15compound 5 μM  <1% <6% NC NC 26 *denotes not compatible with excitationwavelengths and fluorescence detection filters available on unmodifiedcommercially available instruments.

Fluorescence intensity data was accumulated in all-instrument channelswithout any compensation for spectral crossover. S/N was calculated asthe ratio of the mean fluorescence intensity-per cell for labeled cellsdivided by the mean fluorescence intensity per cell for the in labeleddiluent treated controls in the same co-culture. Percent retention wascalculated as % of predicted MFI, where predicted MFI=MFI @T0/foldgrowth at T22-24 (to correct for growth related probe dilution). Percentspectral overlap (Table 3) was estimated as the ratio of MFI for labeledcells in the channel of interest to MFI in the primary channel for agiven probe (channel with maximum S/N ratio).

TABLE 3 Compound (25) (13) (8) FACS Vantage FACS Vantage Channel FACSCalibur SE SE FL2 488 nm 0.001% ND ND excitation; 585/42 BP (MFI = 31)FL3 Calibur: 488 nm  1.6% 0 0 excitation 661/16 BP MFI = (−389) (MFI =0) Vantage 488 nm Excitation; 610/20 BP FL4 Calibur: 635 nm NA* NA NAexcitation; 661/16 BP (MFI = 24430) (MFI = 7180) (MFI = 8087) Vantage647 nm excitation; 740 LP FL5 Vantage only: NA 62.4% 7.6% 647excitation; 740 LP (MFI = 4484) (MFI = 615) *denotes not applicable

EXAMPLE 12 Signal:Noise Estimation for In Vitro Imaging YAC-1 TumorCells Labeled with the FR/NIR Probes

Logarithmically growing YAC-1 cells were labeled with PKH26 or examplecompounds of the present invention at the MTC for each probe. A ZeissAxiophot inverted fluorescence microscope was used at 100× magnificationin combination with a CCD camera and commercially available filtercombinations to image labeled cells or unstained control cells. Forthese preliminary studies, acquisition times were chosen to achievesimilar but sub-saturating signal intensities for all samples analyzed.Filter combinations used for data acquisition Although the off-the-shelffilters available from Chroma Technologies were not optimally matchedwith the excitation and emission spectra of the PTIR probes, good signalintensities were readily obtained for all of the new probes usingrelatively short acquisition times (5-500 msec.). Standard rhodaminefilters (not shown) were used for PKH26 image acquisition, since thesehave a near optimal match to the excitation-emission characteristics ofPKH26. Custom filter sets from Chroma, Technologies, Inc. were asfollows: PKH26-standard rhodamine filter set;

-   -   compound 13—excitation HQ665/45, dichroic Q69OLP, emission        HQ725/50;    -   compound 15—excitation HQ755/75, dichroic Q805LP, emission        HQ860/100;    -   compound 26—excitation HQ775/50, dichroic Q805LP, emission        HQ845/55.        Signal:Noise Estimates for Labeled Cells

Average fluorescence intensity was determined for each of 9cell-associated analysis regions having an area of 44 arbitrary units(lightly shaded circles), avoiding measurements of pixels representingoverlapping cells. Four additional non-cell-associated analysis regions,also with area of 44 arbitrary units (darkly shaded circles, N) wereused to estimate noise level. Mean intensity of all cell associatedregions was calculated (signal, S) as was the mean intensity of allnon-cell associated analysis regions (noise, N). Mean noise values weresimilar for all 4 probes despite the wide range of imaging acquisitiontimes employed, indicating that noise was being averaged over time.Since acquisition times varied widely (5 msec for COMPOUND 13, 50 msecfor PKH26 and COMPOUND 15, 500 msec for COMPOUND 26), comparativeestimates of S/N expected for a standardized 50 msec acquisition timewere calculated as follows:S/N(50 msec)=S/N(observed)×@(50 msec/actual acquisition time in msec).

Despite the fact that spectral characteristics of probe and filters weresignificantly better matched for PKH26 than for the present probes,normalized SN increased as excitation emission characteristics shiftedfrom visible (PKH26) to FR (compound 13) to NIR (compound 15). Withoutbeing bound by theory, the somewhat lower normalized SN observed forcompound 26 may be due to a combination of lower staining concentrationand lower efficiency of the excitation-emission filters.

Authfluorescence:Background Ratio for Unstained Controls

Because measurements at the longer wavelengths are not routinelyperformed and the filters used for the present probes were non-standardcombinations, we also evaluated cell associated autofluorescence andnon-cell associated background using the same filter windows used foranalysis of labeled cell images. However, in this case a constant imageacquisition time of 5000 msec (5 sec) was used for all-filter windows.Reduced autofluorescence signal was seen in the FR (compound 13) and NIR(compound 15 & 26) windows. In addition, background noise was reduced inthe NIR (compound 15 & compound 26 filter windows) compared with thevisible (PKH26) and FR (13).

EXAMPLE 13 Compatibility of Compound 8 and Compound 25 with Three ColorLymphocyte Subset Analysis on a BD FACSCalibur Benchtop ClinicalAnalyzer

Human peripheral mononuclear cells (PBMC) were isolated from the bloodof healthy individuals, labeled with compound 8 or compound 25 atconcentrations similar to those used for proliferation monitoring withPKH26. A variety of monoclonal antibody reagents and fluorochromesuseful for 3-color lymphocyte inimunophenotyping are known in the art.Several of these were evaluated for compatibility with use of the FRprobes as the fourth color. After staining with compounds of the presentinvention, cells were additionally incubated with one of the fluorescentanti-PBMC antibody markers as indicated. Following antibody labeling,flow cytometric analysis was carried out using the BD FACSCaliburclinical analyzer as in Example 10. Post staining recovery was evaluatedby comparing analysis time required to acquire 10,000 CD3+ lymphocytesand viability was assessed as % of lymphocytes able to exclude propidiumiodide (PI).

Referring now to Table 4, there are shown the analyses of the effect ofcompound 8 and compound 25 labeling on three color lymphocyte subsetanalysis.

TABLE 4 Mab- CD8- CD16-PE + CD4-Per CD8- Fluorochrome CD3-FITC CD19-PEcychrome CD56-PE CP cycbrome Membrane probe None 73 3 3 20 49.8 27 1 μMCompound 73 2 3 21 49.4 27 8 2 μM 73 3 4 20 49.9 27 COMPOUND 25 % oftotal lymphocytes positive for indicated monoclonal antibody;representative results from one of three experiments.

EXAMPLE 14 Visualization of FR Labeled Cells by Intact Organ Microscopy

MDA-MB-435s tumor cells were labeled with 5 μM compound 8 as in theabove examples and infused intravenously into a rat. Isolated perfusedintact lung preparations from infused rats were isolated and visualizedusing a BioRad Radiance 2000 confocal microscope. Photographic analysisof the superficial microvessels of an isolated perfused lung preparationenabled detection of areas labeled with compound 8 as red areas whilegreen areas were indicative of cells labeled with CM-fluorescein proteinlabel or autofluorescing endothelial cells. This example demonstratedthe utility of tracking labeled cells in intact organ preparations.

EXAMPLE 15 In Vivo Tracking and Imaging of Tumor Cells Labeled withFR/NIR Probes

It is known in the prior art that NIR labeled antibodies can be attachedto tumor cells and through visualition of the tumor cells with an imageintensified video camera or a cooled CCD camera (46). YAC-1 cells werestained with PKH26, compound 8, compound 13, compound 15, and compound26 at their respective MTC's (10 μM for all but compound 26 at 5 μM).This example demonstrated that bright fluorescence of cells labeled withcompound 13, compound 15 and compound 26 can be detected through theskin of nude mice. 10⁶ YAC-1 cells in 20 μl volumes were injectedsubcutaneously into nude mice and the mice were anesthetized with SodiumPentobarbital (30 Mg/kg) and set over a mobile stage. Excitation wasperformed with an optic fiber illuminator equipped with the appropriateexcitation filter Emission was collected with the aid of a CCD cameraequipped with a Nikon lens through the [KM1] emission filter. Spots ofan absorbing dye were applied to the skin surface in the region ofinjection site 1. At site 1 cells labeled with PKH26 were injected intothe region demarcated: by the red oval and imaged with a standard filterset with 15 seconds exposure. Fluorescence was obliterated in the regionwhere the marker dye was applied, indicating that cells weresubcutaneous. At site 2 cells labeled with compound 13 were injectedinto mouse and imaged with a compound 13 filter set for 6.5 secondsexposure with or without concomitant low intensity backgroundillumination. No PKH26 fluorescence was detected at this site butcompound 13 fluorescence was observed. At site 3 cells labeled withcompound 15 were injected in the mouse and imaged with a filter set for40 seconds exposure. No PKH26 or compound 13 fluorescence was detectedat this site while compound 15 fluorescence was observed. At site 4cells labeled with compound 26 were injected into the region demarcatedby the yellow oval and imaged with a Filter set for 60 seconds exposure.Both compound 15 and compound 26 fluorescence were detected at thissite.

The above description and drawings are only illustrative of a preferredembodiment which achieves the objects, features, and advantages of thepresent invention, and it is not intended that the present invention belimited thereto. Any modifications of the present invention which comewithin the spirit and scope of the following claims is considered partof the present invention.

1. An in vivo method for assessing the shedding rates of mature surfaceepithelial cells of a warm blooded animal comprising the steps of:labeling mature surface epithelial cells at a target site of the animalwith a cyanine dye, and monitoring the site for the presence or absenceof the label following said labeling step, wherein the cyanine dye is acompound having the structural formula:

wherein Z is a biologically compatible counterion.
 2. A compound havingthe structural formula

wherein Z is a biologically compatible counterion.
 3. An in vivo methodfor assessing the shedding rates of mature surface epithelial cells of awarm-blooded animal, comprising the steps of: labeling mature surfaceepithelial cells at a target site of the animal with a cyanine dye;exciting the target site with a light source; and monitoring the sitefor the presence of absence of fluorescence resulting therefrom; whereinthe cyanine dye is a compound having the structural formula:

wherein Z is a biologically active counterion.
 4. The method of claim 3wherein the site is a mucosal surface.
 5. The method of claim 4 whereinthe mucosal surface lines the surface of the gastrointestinal tract, therespiratory tract, or the genitourinary tract.
 6. The method of claim 4wherein the site is a mucosal surface of the stomach.
 7. The method ofclaim 4 wherein the site is a mucosal surface of the colon.
 8. Themethod of claim 3 wherein cell shedding rates are detected by observingchanges in the level of the label at the site at a pre-selected timefollowing said labeling step.
 9. The method of claim 3 wherein theexcitation light has a wavelength of from about 600 nm to about 900 nm.10. The method of claim 3 wherein the epithelial cells are labeled bydirect application of a labeling composition to the site.
 11. A methodfor labeling a cell, comprising the steps of: contacting the cell with acompound having the structural formula:

wherein Z is a biologically compatible counterion.
 12. The methodaccording to claim 11 wherein said labeling composition comprises apharmaceutically acceptable vehicle.